The long term objective of this proposal is to elucidate mechanisms that regulate gene expression post.transcriptionally. This objective will be addressed by examining the translational control of herpes simplex virus type 1 DNA polymerase gene (pol) expression. Pol protein synthesis is repressed at times whenpol mRNA continues to accumulate and data suggest that pol regulation requires viral late gene expression. The Specific Aims of this proposal are: (1) To determine if pol mRNA translational control is mediated by poly(A) tail length or requires expression of the HSV gene, Usll. (2) To identify the mRNA elements required for the regulated pol translation. (3) To identify the trans-acting factor(s) required for pol mRNA accumulation and translational repression. The polyadenylation status of the pol transcript will be assessed by RNase H blotting. The role of the HSV late gene, Usll, in pol regulation will be examined by infecting cells with a HSV Usll null mutant. Usli is the only identified HSV sequence.specific RNA binding protein. The mRNA elements required for pol mRNA translational repression will be identified by transient expression/infection assays using chimeric mRNAs containing discrete po1 mRNA sequences. Identified elements will be analyzed further by mutation/deletion analysis. Viruses containing selected mutations will determine if the mutation disrupts pol regulation within the HSV genome and assess the consequences of pol de.regulation in the viral life cycle. Other viral and/or cellular proteins involved in pol mRNA translational regulation will be determined by electrophoretic mobility shift assays and Uv cross.linking. Potential RNA transacting factors will be identified by probing total RNA fractionated from HSV.infected cells. These experiments should identify po1 rnRNA regulatory elements and the trans.acting factors required for pol mRNA translational control. Understanding pol regulation is clinically germane: HSV is a serious pathogen in jmmunocompromised patients.